Many types of studies require analysis of a large number of genomic regions, with as many as 200,000 target regions analyzed. Normally, each target region is amplified in a separate reaction in an individual sample. The sample requirements and expense of so many separate reactions makes studies with large numbers of genomic targets prohibitive. The complete analysis of complex multi-component systems is frequently beyond the capability of existing methods. Simplification of the system, i.e., selection of the key components and discarding others, allows a useful study to be designed.
In the case of nucleic acid sequencing, isolation of the target sequence is frequently realized by amplification (e.g., by PCR). One solution is to combine the multiple amplification and isolation reactions into a single vessel, i.e., multiplexing. However, two problems arise. First, for PCR, the interaction between the probes generates large numbers of spurious products if multiple probes are combined. Even if this problem is solved, the efficiency of isolation is not uniform, resulting in a large range of concentrations for the selected targets. This concentration range then requires a large dynamic range of sensitivity for the subsequent analysis. For example, a typical DNA chip has a useful sensitivity range of ˜30×. If the PCR amplicon mixture is probed with such a DNA chip, all amplicons below the 30× range could not be detected. Targeted gene sequencing presents a similar problem. A single gene is about 1 part in 105 of a whole human genome, while the exons in one gene are about 2 parts in 106 of a whole genome. Thus, a typical targeted gene study involving 10-500 genes would require the extraction of 100-5000 separate gene fragments per sample. Furthermore, a genomic sample of 3 μg of human DNA contains about 106 copies of the genome, and the range of isolated genes would vary in copy number by 100-1000×. Additionally, some applications, such as heterogyzous genotyping, may require read depths of 20× or more, which translates the whole sample being sequenced to a depth of ˜20,000×, making the cost of the data very high. If all fragments could be extracted in one or a few reactions (i.e., multiplexed) much less sample would be required, resulting in lower reagent consumption and labor costs. However, none of the available mutiplexing methods provide uniform efficiency.
Accordingly, there is a need for methods for isolation of multiple genes with substantially similar representation levels.